1. Field of the Invention
The present invention concerns a new method for the biological preparation of 1,3-propanediol from sucrose, comprising culturing a microorganism genetically modified for the bioproduction of 1,3-propanediol, wherein the microorganism comprises a two-step metabolic pathway for the production of 1,3-propanediol from 4-hydroxy-2-ketobutyrate, comprising a first step of decarboxylation and a second step of reduction, and wherein said microorganism has been modified to be able to use sucrose as sole carbon source.
2. Description of Related Art
Fermentative production of 1,3-propanediol by culturing microorganism producing 1,3-propanediol is known in the art. Methods of production of 1,3-propanediol involving vitamin B12-dependent enzymes have already been described; these methods make the production process very expensive.
There is an ongoing need for alternative solutions to produce 1,3-propanediol with vitamin B12-independent pathway, from renewable sources of carbon. Moreover, there is an ongoing need for the improvement of the overall yield of product being produced, based on the necessary energy for such production. Finally, there is an ongoing need for controlling the level of impurities and by-products, for isolation of the product and its marketing and further use.
1,3-propanediol is mainly produced from glycerol (see the patent application PCT/EP2010/056078) and from glucose via the intermediate glycerol. Since the mondial glycerol stock is limited, there is a need to find other carbohydrates sources.
Carbon sources used in fermentation media generally consist in carbohydrates, mostly derived from plants. Starch is the most abundant storage carbohydrate in plants.
As the cost of the biotechnologically produced commodity chemicals is mainly related to the cost of raw material (i.e. the cost of the fermentation substrate), use of refined sugars is not an economically sustainable choice for industrial scale production. Less expensive substrates are needed that retain a high content of fermentable sugar. In this respect, sucrose coming from the sugar industry represents a good option.
Sucrose is obtained from sugar plants such as sugar beet, sugarcane, sweet sorghum, sugar maple, sugar palms or blue agaves. The different sucrose containing intermediates, products or by-products from the sugar processes (raw juice, thin or clarified juice, thick juice, sucrose syrup, pure sucrose, molasse) may serve as fermentation feedstock.
Two different systems for the uptake and utilization of sucrose in microorganisms have been characterized.
The first one is based on a phosphoenolpyruvate (PEP)-dependent sucrose phosphotransferase system (sucrose PTS) where sucrose is taken up and phosphorylated using phosphoenolpyruvate (PEP) as a donor to yield intracellular sucrose-6-phosphate. Sucrose-6-phosphate is then hydrolysed to D-glucose-6-phosphate and D-fructose by an invertase. D-fructose is further phosphorylated to D-fructose-6-phosphate by an ATP-dependent fructokinase and can then enter the central metabolism. Such a system has been described in several bacterial species, gram-positive as well as gram-negative. Among the Enterobacteriaceae family, more than 90% of wild-type Klebsiella but less than 50% of Escherichia and less than 10% of Salmonella strains are sucrose positive.
A conjugative plasmid pUR400 bearing the genes scrKYABR coding for the sucrose PTS has been isolated from Salmonella (Schmid et al., 1982, Schmid et al., 1988).
A second system called “non-PTS system” was discovered more recently in E. coli EC3132 (Bockmann et al., 1992). This system involves the genes cscBKAR coding for a sucrose:proton symport transport system (CscB), a fructokinase (CscK), an invertase (CscA) and a sucrose-specific repressor (CscR).
Escherichia coli K12 and its derivatives cannot utilize sucrose. However, this ability can be conferred by the transfer of the genes coding for the two previously described systems. This has been demonstrated by transferring the plasmid pUR400 in E. coli K12 (Schmid et al, 1982) or different plasmids (including pKJL101-1) bearing the cscBKAR genes in a sucrose negative strain of E. coli (Jahreis et al., 2002). As for industrial application, tryptophan production from sucrose has been documented in E. coli K12 (Tsunekawa et al., 1992), hydrogen production was shown in E. coli carrying the pUR400 plasmid (Penfold and Macaskie, 2004) and production of different amino-acids by transferring both systems, PTS and non-PTS was reported in patent application EP1149911.
Surprisingly, by combining genetic modifications leading to a sucrose utilization in E. coli strains unable to utilize sucrose, and a specific biosynthetic pathway for 1,3-propanediol, the inventors of the present invention were able to obtain improved yield of 1,3-propanediol production from a renewable source of carbon, sucrose.